Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors–targets of neonicotinoids

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dβ1, and Dβ2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dβ3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dβ3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Neonicotinoid insecticides targeting insect nAChRs are effective, broad-spectrum insecticides [16,[19][20][21][22][23]. Following the discovery of the nitromethylene heterocyclic compound nithiazine, this initial lead compound was modified extensively resulting in 3 generations of commercial neonicotinoids [15]. They exhibit higher affinity for insect over vertebrate nAChRs, thereby resulting in selective toxicity to insects [24,25]. Their high systemic activity in plants has permitted seed treatment which has accelerated their deployment for crop protection. However, potential adverse effects on pollinators such as honeybees, bumblebees and solitary bees are a concern [16,20,26,27], even though reduced numbers of pollinators and other non-target insects are not simply due to the effects of neonicotinoids [16,26]. Adverse effects on aquatic invertebrates and birds are also reported [28,29]. It is vital to understand in detail the mechanism of insect nAChR-neonicotinoid interactions but until recently that was precluded by the difficulty of heterologously expressing robust insect nAChRs.
To begin to address this shortfall in understanding, we first showed that the D. melanogaster Dα3 subunit co-exists with the Dα1, Dα2, Dβ1, and Dβ2 subunits in ejaculatory duct neurons of adult D. melanogaster. Co-expression in X. laevis with the Dα3 subunit increased possible nAChR subtypes from 4 to 12. All the 12 recombinant fruit fly nAChRs will be explored and their sensitivity to the transmitter acetylcholine (ACh), neonicotinoids (imidacloprid, thiacloprid, and clothianidin) and α-bungarotoxin compared. The impact of the Dα1, Dα2, and Dα3 subunits on recombinant nAChR receptor affinity is addressed. The impact of RNAi targeting particular subunits on toxicity to larvae and adult toxicity and behaviour is investigated. Finally, we studied the effects of replacing one of the α subunits in the Dα1/Dα2/Dα3/Dβ1/ Dβ2 nAChRs by either the Dα4 or the Dβ3 subunit on the neonicotinoid actions to address whether such subunit compensation further expands the diversity of neonicotinoid actions in insects.

Results and discussion
We first examined whether the Dα3 subunit is co-expressed with Dα1, Dα2, Dβ1, and Dβ2 subunits, previously shown to be present in ejaculatory neurons of D. melanogaster [30], and if so, how such co-expression influences actions of neonicotinoids in vitro and in vivo. To analyse the expression of Dα3, we stained male ejaculatory neurons with a tyrosine decarboxylase 2 targeting antibody (anti-Tdc2) in the animals expressing GFP under control of Dα3-T2A-Gal4 and found that Dα3 is indeed expressed in the male ejaculatory neurons ( Fig  1A). Another recent study has shown similar findings for the oviduct neurons in female fruit flies [12], suggesting that Dα1, Dα2, Dα3, Dβ1, and Dβ2 subunits can potentially generate diverse heteromeric nAChRs in male and female adult neurons involved in reproductive functions.

PLOS GENETICS
Effects of subunit composition and compensation of fruit fly nicotinic receptors on neonicotinoid actions nAChR was resistant to this neurotoxin [33]. Diverse pharmacology in terms of the sensitivity to α-BTX confirms that 12 distinct, robust, and functional nAChRs results from combinatorial assembly from the five subunits.
We measured agonist activity of imidacloprid, thiacloprid, and clothianidin, in terms of their pEC 50 and I max values for 8 Dα3-containing nAChRs and analysed factors determining them (Fig 3A, Fig C in S1 Text and Table 1). The pEC 50 value for each neonicotinoid relies primarily on the subunit properties (Fig 3B and Table 1, and Tables B and F in S1 Text for ANOVA analysis). For thiacloprid, the affinity for the Dα1/Dα3/Dβ1 nAChR (pEC 50 = 8.07, EC 50 = 8.51 nM) was higher than that for the Dα1/ Dα2/Dβ1 nAChR (pEC 50 = 6.92, EC 50 = 120 nM, Fig 3B, Table 1, and Table D in S1 Text for ANOVA analysis). Inversely, the Dα2 subunit reduced the affinity of neonicotinoids (imidacloprid and clothianidin, Dα2/Dα3/Dβ1 nAChR < Dα3/Dβ1nAChR; thiacloprid, Dα2/Dα3/ Dβ1 nAChR < Dα1/Dα3/Dβ1 nAChR, Tables B, D, and F in S1 Text for ANOVA analysis). Compound properties also contribute to determining the affinity as indicated by the highest pEC 50 values of thiacloprid for most of the nAChRs (Fig 3A and 3B, Table 1, Tables B, D, and F in S1 Text for ANOVA analysis). Such compound factors were more evident in the I max values ( Fig 3C, Table 1, and Tables C, E, and G in S1 Text for ANOVA analysis). For all the nAChRs tested, the order of I max was clothianidin > imidacloprid > thiacloprid, similar to the efficacy order observed in the fruit fly neurons [34], which supports the utility of using the X. laevis oocytes to express nAChRs for the evaluation of neonicotinoid actions in insects.
To clarify the relationship of the nAChR subunits and the neonicotinoids tested with the affinity and efficacy of neonicotinoids for the 12 fruit fly nAChRs (Table 1), we quantitatively analysed the factors governing the variations in the agonist activity indices ( Table 2, and  Table H in S1 Text for parameter and data sets). The adjusted coefficients of Dα1 and Dα2 subunits for affinity were -0.306 and -0.754, respectively ( Table 2), suggesting that both subunits reduced the neonicotinoid affinity, the Dα2 contribution being higher than the Dα1 contribution, while the coefficient of Dα3 was 0.524, indicating that the subunit enhanced affinity ( Table 2). Also, the compound properties underpin the affinity ( Table 2). It was impossible to elucidate the contribution of the Dβ1 subunit since it is common to all the nAChRs being an essential subunit. However, we showed previously that the R81T mutation in the Dβ1 subunit strikingly reduced the affinity and efficacy of the neonicotinoids [30], indicating its critical role in determining neonicotinoid action [16,35,36]. On the other hand, the I max relied mainly on the compound properties even though the values also varied with subunit composition (Fig 2C and Table 2). The highest efficacy of clothianidin probably results from hydrogen bond formation of NH of its guanidine moiety with the backbone carbonyl of the tryptophan in loop B conserved in the insect α subunits [37].
The subunit factors governing variations in neonicotinoid affinity for the various fruit fly nAChRs were derived solely from the multivariate analyses. Therefore, to confirm the results, we performed the chaid (Fig 4A) and lattice (Fig 4B) analyses of the affinity of the neonicotinoids. In the chaid analysis, the Dα1 and Dα2 subunits were negative determinants, whereas the Dα3 subunit was a positive determinant of the affinity, Dα2 being a higher contributor than Dα1 and Dα3 (Fig 4A). Mean pEC 50 of all the neonicotinoids tested for nAChRs without Dα1 and Dα2, but with Dα3 was highest (7.506), indicating that Dα3 is the most critical determinant of high sensitivity for all the neonicotinoids tested of the D. melanogaster nAChRs. In the lattice analysis (Fig  4B), Dα2 subunit was a significant negative factor for the affinity (Table I in S1 Text).
Based on these results, we knocked down Dα1, Dα2, and Dα3 subunit genes by using a panneuronal Gal4 driver (elav-Gal4) and quantified the expression of genes encoding all nAChR subunits in Control (elav-Gal4>w 1118 ) and RNAi animals in both developmental (white prepupae) and adult stages of D. melanogaster (Fig 5A). At the same time, these RNAi animals were used to examine toxicity of imidacloprid, thiacloprid, and clothianidin (Fig 5B). We here focused on Dα1, Dα2, and Dα3 subunits, because these three subunits play critical roles in determining the affinity of the neonicotinoids (Tables 1 and 2). Knockdown of Dα1, Dα2, and Dα3 differentially affected the other subunit gene expression level, depending on stage and sex (Fig 5A). During development, knockdown of each of Dα1, Dα2, and Dα3 hardly affected other subunit gene expression except for Dα2 RNAi, which significantly reduced Dα1 expression. By contrast, knockdown of Dα1 enhanced Dβ3 expression in both males and females and Dα7 expression in adult females. Knockdown of Dα2 reduced Dα1, Dα6, and Dα7 expression in both males and females, and Dβ1 expression in adult males. Furthermore, knockdown of Dα2 enhanced Dβ3 expression in adult males. On the other hand, knockdown of Dα3 reduced Dα1 expression and enhanced Dα2 expression in both males and females while enhancing  Table 2 for the results of multivariate analyses of subunit and ligand factors governing variations in pEC 50  Dβ3 expression in adult females (Fig 5A). These findings indicate that the subunit compensation occurs more frequently in adults than during development. Such subunit compensation can also enhance the inhibitory effect on the climbing behaviour, thereby inducing hypersensitisation to neonicotinoids. Several studies investigated the effects of knocking out nAChR subunit genes in fruit flies on the toxicity of neonicotinoids to larvae or adults and showed that in almost all cases, such mutations resulted in reduced sensitivity to neonicotinoids [38][39][40].
Interestingly, Perry et al. showed that Dα2 knockout enhanced nitenpyram sensitivity in larvae [38]. Also, Chen et al. showed that Dα1/Dβ2 double knockouts reduced imidacloprid resistance level compared to that observed in a Dα1 or a Dα2 single gene knockout in D. melanogaster [41]. Still, the mechanism of these findings is not known. Liu et al. showed a higher Dα3 subunit contribution to the interactions with clothianidin than for other subunits tested in terms of lethal activity [40]. However, no such Dα3 preference for clothianidin was evident in our study. These findings may be attributable, at least in part, to the differences in the nAChR subunits involved in toxicity. Here we show that RNAi of Dα1 and Dα2 reduced toxicity of imidacloprid, thiacloprid, and clothianidin in larvae (Fig 5B), which is attributable to increased non-target/target nAChR ratio. However, as predicted by the multivariate analyses, RNAi of Dα2 led to hypersensitivity to imidacloprid and thiacloprid in adult males and females and to clothianidin in adult males (Fig 5B), which counsels caution in believing that reduction of drug sensitivity generally happens in response to suppressing the primary target proteins. A direct interpretation of such an observation is that Dα2 subunit is the negative factor reducing the affinity of neonicotinoids (Fig 4 and Table 2), hence reduced Dα2 gene expression results in enhanced neonicotinoid sensitivity. The qRT-PCR data (Fig 5A) revealed that in response to RNAi of Dα2, expression of genes encoding Dα5, Dα6, and Dα7 subunits, of which the Dα5 and Dα6 subunits form low imidacloprid-sensitive nAChRs [42], was reduced, offering another explanation for the enhanced toxicity of neonicotinoids. Reduced toxicity by knockdown of Dα2 and concomitant reduced Dβ1 expression was also observed in adult males (Fig  5A), which can reduce numbers of nAChRs with neonicotinoid sensitivity since the Dβ1 subunit is essential for functional expression (Fig 1 and Table 1). As such, subunit compensation in response to the knockdown of Dα1, Dα2, and Dα3 varies with developmental stages and sexes as well as the primary target of RNAi, resulting in diverse neonicotinoid actions.
In conclusion, by studying 18 subunit combinations of subunits Dα1, Dα2, Dα3, Dα4, Dβ1, Dβ2, and Dβ3, we have found that imidacloprid, thiacloprid and clothianidin can interact with a broad range of D. melanogaster nAChRs formed not only by the Dα1, Dα2, Dα3, Dβ1, and Dβ2 subunits, but also by the Dα4 and Dβ3 subunits, which has not been described to the best of our knowledge. Although co-expression of these subunits does not necessary prove that they co-assemble to form functional nAChRs in neurons, it is clear that the three neonicotinoids exhibited diverse agonist actions on the 18 nAChRs tested, the outcome depending on both the compound as well as subunit composition. Notably, the Dα1, Dα2, Dα3, Dβ1, and Dβ2 subunits co-localise in organs underlying mating and egg laying, predicting that modulation of the nAChRs consisting of these subunits will affect the number of offspring. In future, it will be of considerable interest to test this hypothesis. If such actions are confirmed, not only for the fruit flies, but also for other insect species such as pollinators and disease vectors, this will counsel further caution in identifying target receptor subtypes simply in terms of reduced neonicotinoid sensitivity resulting only from gene disruption or suppression experiments.

Ethics statement
Oocytes at stage V or VI of development were removed under anesthetic (0.3 g L -1 benzocaine) from adult female X. laevis according to the U.K. Animals (Scientific Procedures) Act, 1986. Care was always taken to minimise the number of animals used in experiments.

Generation of Dα3-knock-in T2A-Gal4 strain
The Gal4 knock-in D. melanogaster flies were generated by CRISPR/Cas9-mediated homologous recombination. A targeting vector was designed such that the T2A-Gal4 [45] is inserted in frame with the last intracellular region of the protein. The targeting vector and a gRNA expression vector that cuts near the target site were co-injected into fertilised eggs maternally expressing Cas9 protein. The flanking sequences of the insertion are: 5´-GAAAGAGGACTG GAAGTACGTGGCCATG/GTGCTCGATCGCCTGTTCCTGTGGATCTTCACAATAGC-3( The site of integration is indicated by a slash, The 20-bp gene-specific sequence of the gRNA is underlined.) Immunostaining D. melanogaster male reproductive systems were dissected in Grace's Insect Medium, supplemented (Thermo Fisher Scientific, USA, #11605094) and fixed in 4% paraformaldehyde in Grace's medium for 30-60 min at room temperature (RT). The fixed samples were washed three times in phosphate-buffered saline (PBS) supplemented with 0.1% Triton X-100 (Nacalai Tesque, #12967-45). After washing, samples were blocked in the blocking solution (PBS with 0.1% Triton X-100 and 2% bovine serum albumin (MilliporeSigma, #A9647) for 1 h at RT then incubated with a primary antibody in the blocking solution at 4˚C overnight. The primary antibodies used in this study were mouse anti-GFP monoclonal antibody (clone GFP-20; MilliporeSigma G6539; 1:1000) and rabbit anti-Tdc2 antibody (Abcam ab128225; 1:1000) [46]. Fluorophore (Alexa Fluor 488, or 546)-conjugated secondary antibodies (Thermo Fisher Scientific, #A11001, #A32732) were used at a 1:200 dilution and incubated for 2 h at RT in the blocking solution. After washing, all samples were mounted in FluorSave reagent (Millipore-Sigma, #345789). Samples were visualised on an LSM 700 confocal microscope (Zeiss, Germany). Images were processed using the ImageJ package [47].

Voltage-clamp electrophysiology
Each defolliculated X. laevis oocyte was secured in a Perspex recording chamber and perfused with the standard oocyte saline (SOS) containing 0.5 μM atropine (SOSA) at a flow rate of 7−10 mL/min [48]. Two glass electrodes filled with 2 M KCl were impaled into each oocyte and the membrane potential was clamped at -100 mV. ACh and α-BTX (Alamone Labs, Israel, #B-100) were dissolved directly in SOSA, while test solutions of the neonicotinoids were diluted to the final concentration from DMSO stock solutions. DMSO at 1% (v/v) or lower had no effect on the responses to neonicotinoids or other ligands tested. ACh and neonicotinoids were applied for 5 s successively at 3 min intervals. α-BTX was tested as previously described in the literature [33]. The peak amplitude of the response was measured and analysed by pCLAMP (Molecular Devices, USA). The agonist response data were normalised to the maximum response to ACh at concentrations at which the response amplitude attained plateau and fitted by non-linear regression using Prism (GraphPad Software, USA), according to the following equation. 50 is the half maximal concentration (M), I max is the maximum normalised response and n H is the Hill coefficient.

Total RNA extraction and quantitative reverse transcription (qRT)-PCR
Animals were collected in 1.5 ml tubes and immediately flash-frozen in liquid nitrogen. Total RNA from white prepupa (0 hour after puparium formation) or adults (3 days after eclosion) was extracted using RNAiso Plus (Takara Bio, Japan, #9109) according to the manufacturer's instructions. cDNA was generated from purified total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan, #FSQ-301). qRT-PCR was performed on the Thermal Cycler Dice TP800 system (Takara Bio) using THUNDERBIRD Next SYBR qPCR Mix (Takara Bio, #QPX-201). For absolute quantification of mRNAs, serial dilutions of plasmids containing coding sequences of the target genes or rp49 were used for standards. After the molar amounts were calculated, transcript levels of the target mRNA were normalised to rp49 levels in the same samples. The primers used are listed in Table K in S1 Text. The primers to detect rp49 levels are as previously reported [49].

Adult climbing assay
Adult D. melanogaster flies were collected within a day following eclosion and placed in normal fly food (less than 30 flies/vial). Flies were transferred daily to new fly food. 2-5 days after eclosion, flies were briefly anesthetised with CO 2 , and the sexes were separated and sorted into fly vials containing 1.0% agar food for starvation (10 flies/vial). After 16 h starvation, flies were transferred to vials containing neonicotinoid-containing food without anesthesia and cultured for 6 h (10 flies/vial). Neonicotinoid-containing foods were prepared by mixing 10 μL of diluted neonicotinoids dissolved in DMSO with 990 μL of a solution containing 1% agar and 5% sucrose for each vial. After 6 h cultured in vials containing neonicotinoid-containing food, flies were gently tapped down to the surface of the food, and flies that climbed within 20 s after tapping were recorded by a video camera (GZ-F270-W, JVCKENWOOD, Japan). The maximum climbing heights of the flies within 20 s after tapping were measured using ImageJ1.53v (National Institute of Health, USA). Since the height from the surface of the food to the vial top is 8 cm, the maximum climbing height is 8 cm.

Reproducibility of data
At least two authors participated independently in measuring data to confirm reproducibility of the results. For electrophysiology, five oocytes from at least two frogs were used to determine the agonist activity of each ligand at each concentration.

Statistical analyses
Prism software was employed for the statistical analyses. The peak current amplitude of the agonist actions of ACh at 100 μM was compared between the nAChRs by Kruskal-Wallis test. One-way ANOVA was used to analyse the differences of the ligand agonist activity in terms of pEC 50 on the various nAChRs expressed in X. laevis as well as data obtained with D. melanogaster larvae and adults.

Multiple variate analysis
The multiple variate analysis was conducted with python to examine if D. melanogaster nAChR subunits and compounds contribute significantly to the agonist activity in terms of pEC 50 and I max . We used a dataset including 48 samples (Table H in S1 Text). Objective variables are pEC 50 and Imax and explanatory variables are subunits (Dα1, Dα2, Dα3, and Dβ2) and compounds (ACh, imidacloprid, thiacloprid, and clothianidin). Data for ACh were used as references when calculating the subunit and compound factors governing the variations in the agonist activity indices.

Chaid analysis
Chaid analysis was conducted with python to examine if the nAChR subunits contribute significantly to the agonist activity in pEC 50 . Parameter max depth was set as 4. Objective variable is pEC 50 and explanatory variables are subunits (Dα1, Dα2, Dα3, and Dβ2) and compounds (ACh, imidacloprid, thiacloprid, and clothianidin).

Lattice visualization
The lattice visualization was used to observe the positive contribution to pEC 50 of adding each subunit (Dα1, Dα2, Dα3, Dβ1, and Dβ2). The presence or absence of subunits forms the powerset with lattice structure with respect to the inclusion order. The data are grouped by differences between two sets of subunits and denoted by "+<subunit name>" on each edge. The color bar indicates ΔpEC 50 , the value obtained by subtracting pEC 50 for smaller nAChR subunit set from that for larger nAChR subunit set. The significance of the ΔpEC 50 values was analysed by the 95% confidence interval.